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Supplemental Information

Development of a Ratiometric Two-photon Fluorescent Probe for Imaging of

Hydrogen Peroxide in Ischemic Brain Injury

Baoping Zhai,a* Wei Hu,b Ruilin Hao,a Wenjing Ni,a Zhihong Liub*

a Department of Chemistry, Xinzhou Teachers University, Xinzhou, Shanxi 034000, China.

b Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry

and Molecular Sciences, Wuhan University, Wuhan 430072, China.

* Corresponding author.

Tel: +86 27 87217886;

E-mail address: zhhliu@whu.edu.cn

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2019

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Materials and apparatus.

Scheme S1. Synthesis route of FH2O2. Reagents and conditions: a), acetyl chloride,

AlCl3, Dry DCM, 0 °C to r. t. 3 h; b), methylamine aqueous solution, copper powder,

DMSO, 140 °C, 2 day; c), triphosgene, Na2CO3, methylbenzene, r.t., 10 h; d),

pyridine, anhydrous CH2Cl2, r. t. 12 h, under nitrogen atmosphere.

Unless otherwise stated, all solvents and reagents were purchased from

commercial suppliers and used as received without further purification. All aqueous

solutions were prepared in ultrapure water with a resistivity of 18.25 MΩ cm

(purified by Milli-Q system supplied by Millipore). MS analysis was performed

on a liquid chromatograph (Agilent 1200, America) connected to a quadrupole

time-off light mass spectrometer (Q-TOF MS, Agilent 6520, America).

Absorption spectra were recorded on an UV−vis spectrophotometer (Shimadzu

UV-2550, Japan), and one-photon fluorescence spectra were obtained with a

fluorimeter (Shimadzu RF-5301 PC, Japan). Two-photon fluorescence spectra

were excited by a mode-locked Tisapphire femtosecond pulsed laser

(Chameleon Ultra I, Coherent, America) and recorded with a DCS200PC

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photon counting with Omno-λ5008 monochromator (Zolix, China). Two-

photon microscopy images were collected from a spectral confocal and

multiphoton microscope (Carl Zeiss, LSM 780 NLO, Germany) with a mode-

locked titanium-sapphire laser source (Mai Tai HP, Spectra Physics, America).

Synthesis route of FH2O2 was depicted in Scheme S1 and the synthesis of

compound 1 and 3 was referred to the literature.1, 2

Synthesis of FH2O2

2 mL Pyridine was added to a stirred solution of compound 1 (662.5 mg,

2.5 mmol) and compound 3 (1.09 g, 3.7 mmol) in 20 mL dry methylene

dichloride and the reaction mixture was stirred at room temperature for 12 h

under nitrogen atmosphere. The solvent was removed in vacuo and extracted by

methylene dichloride. The organic layer was dried with anhydrous Na2SO4.

Then the solvent was removed under reduced pressure, and the crude product

was purified by silica gel column chromatography using petroleum ether: ethyl

acetate (v:v = 6 : 1) and gave a white solid. (787 mg, 60% yield). 1H NMR (400

MHz, CDCl3) 8.13 (1 H, d, J = 0.9), 7.98 (1 H, d, J = 1.6), 7.97 – 7.91 (2 H, m),

7.62 (3 H, dd, J = 8.0, 4.8), 7.36 – 7.31 (3 H, m), 5.24 (2 H, s), 3.32 (3 H, s),

2.64 (3 H, s), 1.47 (6 H, s), 1.28 (12 H, s). 13C NMR (400 MHz, CDCl3) δ

198.07, 155.58, 155.38, 154.03, 143.49, 135.96, 134.99, 128.41, 126.87,

122.38, 121.24, 119.79, 83.85, 77.26, 67.34, 47.16, 38.03, 26.91, 24.86. MS:

calcd for C32H36BNO5 [M + H] + 526.3 found 526.3.

General procedure for hydrogen peroxide detection

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Unless otherwise stated, all the fluorescence measurements were made in 10

mM PBS (pH 7.4) according to the following procedure. In a 2mL tube, 450 μL

of PBS and 50 μL FH2O2 were mixed, and then add an appropriate volume of

hydrogen peroxide sample solution. After incubation at 37 °C for 40 min in a

thermostat, a 3 mL portion of the reaction solution was transferred to a quartz

cell of 1 cm optical length to measure absorbance or fluorescence with λex/em =

358/562 nm and excitation slit width of 3 nm and emission slit widths of 5 nm.

For the selectivity assay, Superoxide (O2∙-) was created by the enzymatic

reaction of xanthine/xanthineoxidase (XA/XO; 6.0 μM/3 mU) at 25 °C for 5

min.3, 4 ·OH was generated by Fenton reaction between Fe2+ (EDTA) and H2O2

quantitatively, and Fe2+ (EDTA) concentrations represented ·OH

concentrations.5 The ONOO- source was the donor 3-Morpholinosydnonimine

hydrochloride (SIN-1, 200 μmol/mL).6 NO was generated in form of 3-

(Aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5, 100

μM/mL).7 OCl- was standardized at pH 12 (ε292 nm = 350 M-1cm-1).8 H2O2 was

determined at 240 nm (ε240 nm = 43.6 M-1cm-1). 1O2 was generated from 3,3'-

(naphthalene-1,4-diyl)dipropionic acid.9 All the reagents were obtained from

Aladdin (USA). All other chemicals were from commercial sources and of

analytical reagent grade, unless indicated otherwise.

Measurement of Fluorescence Quantum Yield.

The fluorescence quantum yield was determined by using quinine sulfate in

0.1 N sulfuric acid (Φ=0.55). The fluorescence quantum yield was calculated

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according to the following equation:

Φs = Φr(ArIsns2)/(AsIrnr

2) (A≤0.05)

The subscripts s and r represent the sample and the reference molecule

respectively. Φ is the fluorescence quantum yield. A is the absorbance of

molecules that is controlled below 0.05 for both molecules in the same

wavelength. I means the integrated emission area and n is the refractive index

of the solvent.

Measurement of Two-Photon Cross-Section.

The two-photon cross section (δ) was determined by using the femto second

(fs) fluorescence measurement technique as described. 1 and FH2O2 were

dissolved in PBS buffer (10mM, pH 7.4, containing 0.9% NaCl), The

fluorescence intensity was measured at 710-880 nm by using rhodamine B

(dissolved in methanol) as the reference. The two-photon properties in this

reference have been well characterized. The intensities of the two-photon-

induced fluorescence spectra of the reference and sample emitted at the same

excitation wavelength were determined. The two-photon cross section was

calculated by using δ = δr(SsΦrϕrcr)/(SrΦsϕscs), where the subscripts s and r

stand for the sample and reference, respectively. The fluorescence intensity was

denoted as S. Φ is the fluorescence quantum yield and ϕ is the overall

fluorescence collection efficiency of the experimental apparatus, which can be

approximated by the refractive index of the solvent. The concentration of the

solution was denoted as c. δ is for two-photon absorption.

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Determination of the detection limit

The detection limit was calculated based on the fluorescence titration.10, 11

The fluorescence emission spectrum of FH2O2 was measurement 11 times

without adding H2O2 and the standard deviation of blank measurement was

achieved. The fluorescence intensity at 560 nm was plotted against the

concentration of H2O2. So the detection limit was calculated with the following

equation:

Detection limit = 3σ/k

Where σ is the standard deviation of blank measurement and k is the slope

between the fluorescence intensity versus H2O2 concentration.

DFT calculations.

To describe the ground state and singlet excited state of the probe, DFT

theoretical calculations were performed. All the calculations were carried out

using the Gaussian 09 program package.12 All the geometries of TBET system

were optimized at B3LYP/6-31+G(d) level using a CPCM solvation model

with water as the solvent. The molecular orbital (MO) plots and MO energy

levels were computed at the same level of theory.

Cell Culture

Bv-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM,

Thermo Scientific) supplemented with 1 % penicillin/streptomycin and 10 % fetal

bovine serum (FBS), and incubated in an atmosphere of 5/95 (v/v) of CO2/air at 37 °C.

Cells were passed and plated into glass bottom cell culture dishes (NEST) in

the day before imaging. For probe loading, the cells were washed with

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phosphate buffer and then interacted with 10 μM FH2O2 (containing 1 %

DMSO in serum-free DMEM) for 30 min at 37 °C. After washing with

phosphate buffer twice, the cells were then incubated into a phosphate buffer

solution containing for 2 h.

Cytotoxicity Study of FH2O2 for Cells.

MTT test was performed according to the reported protocol with a minor

change. Bv-2 cells were seeded in 96-well plates and incubated with different

concentrations of FH2O2 (0, 5, 10, 15 and 20 μM, containing 1 % DMSO in

100 μL DMEM). The FH2O2 labelled bv-2 cells were incubated in an

atmosphere of 5/95 (v/v) of CO2/air at 37 °C for 24 h. Subsequently, 20 μL 5.0

mg/mL MTT solution was added to each well. Followed by incubation for 4 h

under the same conditions. 100 μL supernatant was removed and 150 μL

DMSO was added. After shaking for 10 min, the absorbance at 490 nm was

measured by microplate reader (Synergy 2. BioTek Instruments Inc.). Cell

survival rate was calculated by A/A0 × 100 % (A and A0 are the absorbance of

the FH2O2 labelled group and the control group, respectively).

Fluorescence Microscopy Imaging.

Bv-2 cells were washed twice with PBS and then cultured with DMEM

without glucose that had been progressed with 95% N2/5% CO2 for 10 min to

remove residual oxygen. bv-2 cells were passed and dispersed on 18 mm glass

coverslips at 37 °C, 5% CO2 1 day before imaging. Then cells were incubated

with 10 μM FH2O2 for 40 min. The medium was removed and cells were

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washed with PBS (10 mM, pH 7.4) twice. Two-photon microscopic imaging of

bv-2 cells which were labelled with 10 μM FH2O2 were performed on a

spectral confocal and multiphoton microscopes (Carl Zeiss, LSM 780 NLO)

with a 40 × water objective, and a numerical aperture (NA = 1.0). The two-

photon fluorescence microscopy images were obtained by exciting FH2O2 with

a mode-locked titanium–sapphire laser source (Mai Tai HP, Spectra Physics, 80

MHz pulse frequency, 100 fs pulse width) set at the wavelength of 750 nm. All

the images were separately collected in the emission wavelength range of 400-

480 nm and 570-670 nm. The internal PMTs were used to collect the signals in

an 8 bit unsigned 1024 × 1024 pixels at a scan speed of 1.58 s per pixel.

Western blotting.

Bv-2 cells were lysed with NP-40 lysis buffer (1.0% NP-40, 150mM NaCl,

50mM Tris-HCl, pH 8.0). The protein were separated by 10% sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred

onto nitrocellulose membrane. To block nonspecific antibody binding, the

membrane was treated in 5% nonfat milk (dissolved in TBS) for 30 min. Next,

the membrane was incubated with the following rabbit antibodies were

purchased from Cell Signaling Technology (β-actin: #4970, 1:1000; LC3-II:

#2775S, 1:1000).

Statistical Analysis.

Statistical Product and Service Solutions (SPSS) software 19.0 was used for

the statistical analysis. The error bars shown in the figures represented the mean

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± s.d. Differences were determined with a one-way analysis of variance

(ANOVA) followed by LSD test. Statistical significance was assigned at *P <

0.05, **P < 0.01 and ***P < 0.001. Sample size was chosen empirically based

on our previous experiences and pre-test results. No statistical method was used

to predetermine sample size and no data were excluded. The numbers of

animals or samples in every group were described in the corresponding figure

legends. The distributions of the data were normal. All experiments were done

with at least three biological replicates. Experimental groups were balanced in

terms of animal age, sex and weight. Animals were all caged together and

treated in the same way. Appropriate tests were chosen according to the data

distribution. Variance was comparable between groups in experiments

described throughout the manuscript.

In vivo imaging studies.

All animal procedures were performed in accordance with the Guidelines

for Care and Use of Laboratory Animals of Hunan University, and experiments

were approved by the Animal Ethics Committee of the College of Biology

(Hunan University). All BALB/c mice (age 4 weeks, weight 17–20 g) were

operated upon in accordance with institutional ethics committee regulations and

guidelines on animal welfare. The aforementioned animals were randomly

divided into control (n = 3), 24-h model (n = 3) and 48-h model (n = 3) groups.

The model groups were anesthetized with isoflurane 7 days after birth,

disinfected, and a median longitudinal incision was made under the operation

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microscope. The common carotid artery was carefully separated and double-

ligated with 6-0 silk thread and disconnected, and the skin incision was sutured.

After 2 h of postoperative recovery, the test was placed in the incubator at 37°C

for 2 h with 8% oxygen in a 92% nitrogen hypoxia chamber. After performing

anesthesia on the control group, only the median neck incision was made

followed by suturing of the incision. After a complete recovery, food and water

were again provided to the animals ad libitum. The local environmental

temperature was maintained at 35°C–37°C during surgery, hypoxia and

resuscitation. Following the hypoxic–ischemic process, abdominal injection of

200 μL of 1.0 mM probe FH2O2 was administered, animals were fasted for 18

h and then decapitated, then the brain tissue was immediately removed, washed

in ice-cold normal saline, and cut into 300-μm section. Sections were washed

with PBS and two-photon tissue fluorescence imaging was immediately

performed.

Glutathione S-transferase (GST) activity assay.

According to the 1-chloro-2,4-dinitrobenzene (CDNB) colorimetric method

as described by Habig, the principle is as follows: GST has the ability to

catalyze the binding of reduced glutathione (GSH) to CDNB, the peak

wavelength of light absorption of the bound product is at a wavelength of 340

nm, and the activity unit of GST can be calculated by measuring the absorbance

at this wavelength. The reaction formula is as follows:

C6H3(NO2)2Cl + GSH → C6H3(NO2)2SG + H+ + Cl−

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The determination procedure involves addition of 5 mL of 0.1 mol/L phosphate

buffer (pH 6.5), then 500 μL of 1 mmol/L GSH, followed by 500 μL crude

enzyme dilution (diluted 10 times), and 500 μL of 1 mmol/L CDNB. After

accurately reacting for 1 min in a 30°C water bath, the tube was immediately

placed in a 60°C water bath to terminate the reaction for 5 min, and after

removal absorbance (A340)0 was measured at room temperature for at least 10

min. The absorbance (A340)1 of the non-enzymatic reaction was measured by

the same procedure using physiological saline instead of the enzyme solution.

Absorbance was measured with a Japanese Hitachir UV-3010 ultraviolet

spectrophotometer.

Calculation method: GST (µmol·min−1·mg−1) = ∆A·ɛ−1·Cp−1·2.6 × 108 where

∆A = (A340)0 − (A340)1, ɛ: molar absorption coefficient of the product at 340

nm is 9.6 × 103 M−1·cm−1, and Cp: protein content (mg/mL).

Glutathione reductase (GRed) activity assay.

For the glutathione reductase activity assay, glutathione peroxidase reduces

lipid peroxide to oxidize reduced glutathione to its oxidized form, while

oxidized glutathione can be reduced by glutathione reductase in coenzyme II in

the presence of a reduced form. The reduced coenzyme can be produced by a

reductive enzyme II (NADPH regenerating enzymes; NRE) such as glucose-6-

phosphate dehydrogenase, 6-phosphate gluconate dehydrogenase, or malic

enzyme. Reduced coenzyme II-dependent isocitrate dehydrogenase and

nicotinamide dinucleotide transhydrogenase are provided.

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The specific measurement procedure is as follows: 4 mL of 0.1 mol/L

phosphate buffer (pH 7.0) containing 1 mM EDTA, 400 μL enzyme dilution,

100 μL of 1 mM reduced coenzyme II, and 500 μL of sample is added to the

test tube. Then 1 mM oxidized glutathione was added, and the final

concentrations of reduced coenzyme II and oxidized glutathione were 0.2 mM

and 1 mM, respectively. After reacting for 5 min in a 37°C water bath,

absorbance (A340)0 was measured at 340 nm, and the non-enzymatic reaction

absorbance (A340)1 was measured by replacing the enzyme solution with an

equal amount of physiological saline (measured in 15 min).

Calculation formula: GRed (µmol·min–1·mg–1) = ∆A·ɛ–1·Cp–1·2.6 × 108 where

∆A = (A340)0 − (A340)1, ɛ: molar absorption coefficient of the product at 340

nm is 9.6 × 103 M–1·cm–1, and Cp: protein content (mg/mL).

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Figure S1 1H-NMR spectrum of FH2O2 (400 MHz, CDCl3)

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Figure S2 13C-NMR spectrum of FH2O2 (100 MHz, CDCl3)

Figure S3 MS spectrum of FH2O2

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Figure S4 MS traces of (A) FH2O2, (B) the product of FH2O2-H2O2 reaction and (C)

the product of FH2O2-ONOO- reaction.

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Figure S5 Effects of pH on the fluorescence (λex/em = 410/560 nm) of FH2O2 (10 μM)

reacting with hydrogen peroxide (200 μM).

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Figure S6. Cell survival rate of control groups (without FH2O2) and experimental

group (with 5, 10, 15, 20 μM of FH2O2). All groups contain 1 % DMSO in

100 μL DMEM)

Figure S7. (A) TPM images of HeLa cells labeled with 200 μM hydrogen peroxide

for 48 h and further incubated with FH2O2 for 30 min separately. (B) Two-

photon fluorescence intensity from circle a-f as a function of time. The two-

photon fluorescence intensity was collected with 15 sec intervals for the

duration of 25 min under xyt mode. Scale bar: 20 μm

Figure S8. TP fluorescence images of HeLa cells labelled with 10 μM FH2O2 for 40

min at 37 °C and further incubated with 0 (a-d), 100 μM (e-h), 200 μM (i-l) H2O2 for

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30 min. Images were obtained by being excited with 750 nm laser light, and the

emissions were collected individually in blue channel (400-480 nm, b, f, j) and yellow

channel (570-670 nm, c, g, k). The fluorescence intensity ratio of the two channels

(Fyellow/Fblue, d, h, l), which was reflected as colour bar processed by Image-Pro Plus

6.0. Cells shown are replicate images from replicate experiments (n=3). Scale bar: 20

μm.

Figure S9. TP fluorescence images of HeLa cells treated with 0 (a-d), 1 (e-h) and 2

μg/mL (i-l) PMA and further being incubated 10 μM FH2O2 for 40 min at 37 °C.

Images were obtained by being excited with 750 nm laser light, and the emissions

were collected individually in blue channel (400-480 nm, b, f, j) and yellow channel

(570-670 nm, c, g, k). The fluorescence intensity ratio of the two channels (Fyellow/Fblue,

d, h, l), which was reflected as color bar processed by Image-Pro Plus 6.0. Cells

shown are replicate images from replicate experiments (n=3). Scale bar: 20 μm.

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Figure S10. TP fluorescence images of FH2O2 labelled HeLa cells preteated with

PMA and H2O2 inhibits. HeLa cells were incubated with untreated (a-d), 10 μM DPI

(e-h) and 10 mM NAC (i-l) for 1 h, and then incubated 1μg/mL PMA for 1 h and

further being incubated 10 μM FH2O2for 30 min at 37 °C. Images were obtained by

being excited with 750 nm laser light, and the emissions were collected individually

in blue channel (400-480 nm, b, f, j) and yellow channel (570-670 nm, c, g, k). The

fluorescence intensity ratio of the two channels (Fyellow/Fblue, d, h, l), which was

reflected as color bar processed by Image-Pro Plus 6.0. Cells shown are replicate

images from replicate experiments (n=3). Scale bar: 20 μm.

Figure S11. Depth fluorescence images of FH2O2 (200 μM) in mouse Brain tissues.

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λex = 750 nm, λem = 570–670 nm. Scale bar: 100 m.

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